Water buffalo derived peptide antibiotic therapies

ABSTRACT

The present disclosure relates to antimicrobial agents and methods of using such agents. The disclosure includes antimicrobial agents having broad spectrum antimicrobial activity, nucleic acids and amino acid sequences encoding such antimicrobial agents, as well as methods of using the antimicrobial agents. The antimicrobial agents of the disclosure may be used to reduce survival of a microbe, as an antimicrobial therapeutic, in microbial treatment protocols, and in research, as well as other uses related to reducing microbe survival. In addition, the disclosure also includes compositions, as well as articles of manufacture, that comprise a broad spectrum antimicrobial agent.

FIELD OF THE INVENTION

The present disclosure relates to antimicrobial agents and methods ofusing. More particularly, the disclosure relates to antimicrobialpeptides and therapeutic uses thereof.

SEQUENCE LISTING

This application contains a sequence listing in paper format and incomputer readable format, the teachings and content of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Antimicrobial resistance is reducing the availability of effectiveantimicrobial treatments worldwide. Resistant organisms, includingbacteria, fungi, viruses, and parasites, are able to withstand attack byantimicrobial medicines, so that standard treatments become ineffective.Infections by such resistant organisms persist increasing the risk ofspreading to others. The evolution of resistant strains of organisms isa natural phenomenon that occurs when microorganisms are exposed toantimicrobial drugs, and resistant traits can be exchanged betweencertain types of bacteria. The misuse of antimicrobial medicines alsoaccelerates the emergence of resistant organisms.

With the decrease in effective antimicrobial treatments due to theemergence of resistant organisms, new antimicrobial therapeutics areneeded. The number of new antimicrobial therapies developed and approvedhas steadily decreased in the past three decades, leaving even feweroptions to treat resistant organisms.

Accordingly, a need exists for antimicrobial treatments and therapeuticshaving broad spectrum antimicrobial activity. Moreover, a need existsfor effectively inhibiting the growth and spread of harmfulmicroorganisms.

SUMMARY OF THE INVENTION

The present disclosure provides antimicrobial agents having broadspectrum antimicrobial activity and methods of inhibiting microorganismgrowth. In particular, the present disclosure is directed to peptideshaving antimicrobial activity and methods of using the peptides toinhibit microorganisms.

The disclosure provides synthetic antimicrobial agents having at leasttwo 14 amino acid peptide sequences operably linked. The amino acidpeptide sequence is generally the sequence of formula 1(Gly-X₁-X₂-X₃-X₁-X₁-X₁-Arg-X₄-X₁-X₅-X₆-X₆-Gly) where X₁ is selected fromthe group of Leu or Ile; X₂ is selected from the group of Ala, Val, Leu,Ile, or Pro; X₃ is selected from the group of Arg or Trp; X₄ is selectedfrom the group of Trp, Ile, or Leu; X₅ is selected from the group of Pheor Trp; and, X₆ is selected from the group of Phe, Trp, or Arg. In someaspects, the amino acid peptide sequence has at least 70% sequenceidentity to a sequence provided in SEQ ID NO: 3-33. In some aspects, theamino acid peptide sequence has at least 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least onesequence provided in SEQ ID NO: 3-33. Preferably, the amino acid peptidesequence has at least 85% sequence identity to SEQ ID NO: 8.

In some aspects, the antimicrobial agent disclosed includes a firstsynthetic amino acid sequence and a second synthetic amino acid sequenceoperably linked. Such second synthetic amino acid sequence, like thefirst synthetic amino acid sequence, is at least a 14 amino acid peptidesequence. Generally, the second synthetic amino acid sequence also has asequence of formula 1 described above. In some aspects, the secondsynthetic amino acid sequence is identical to the first synthetic aminoacid sequence, creating a homodimer. In other aspects, the secondsynthetic amino acid sequence is not identical to the first syntheticamino acid sequence, creating a heterodimer. In some aspects, theantimicrobial agent includes a sequence having at least 70% sequenceidentity to a sequence provided in SEQ ID NO: 40-4101. In some aspects,the antimicrobial agent includes a sequence having at least 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto at least one sequence provided in SEQ ID NO: 40-4101. Preferably, theantimicrobial agent includes a sequence having at least 85% sequenceidentity to SEQ ID NO: 40.

In some aspects, the antimicrobial agent of the disclosure includes alinker sequence that operably connects the first synthetic amino acidsequence to the second synthetic amino acid sequence. Suitable linkersequences further enhance kinetics of antimicrobial activity of theantimicrobial agent. In some aspects, the linker sequence is tailored toenhance solubility in polar environments. In some aspects, the linkersequence is tailored to enhance solubility in lipophilic environments.In some aspects, the linker sequence is tailored to enhance chemicalstability in a variety of chemical environments. Suitable linkersequences may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, or more amino acids in length. Preferably, the linkersequence is about 5, 6, 7, 8, 9, or 10 amino acids in length. Morepreferably, the linker sequence is about 5 to 8 amino acids in length.Suitable linker sequence may include any amino acid in each position, solong as the antimicrobial activity of the antimicrobial agent is notdisrupted. Such linker sequences include those that permit antiparallelalignment of two distinct helices formed by the first synthetic andsecond synthetic amino acid sequences. Such linker sequences alsoinclude those that enhance antimicrobial activity of the first syntheticand second synthetic amino acid sequences. By way of example, withoutlimitation, the first amino acid position of the linker sequence may beproductively occupied with an anionic residue such as aspartate orglutamate, which may enhance antimicrobial activity by stabilizing anactive conformation. Also, by way of example, without limitation, thelinker sequence may include 5 to 8 asparagine residues to enhancesolubility in polar environments while not disturbing the activeconformation incumbent in the remainder of the molecule. Alternatively,the linker sequence may include 5 to 8 glycine residues to enhancesolubility in polar environments while not disturbing the activeconformation incumbent in the remainder of the molecule. In otheraspects, the linker sequence may be of a heterogeneous form, composed ofabout 5 to about 8 residues selected from the following amino acids,including, asparagine, glutamine, glycine, histidine, serine, threonine,and combinations thereof. Suitable heterogeneous formulations may beselected to enhance solubility in polar environments while notdisturbing the active conformation incumbent in the remainder of themolecule.

In some aspects, the linker sequence is generally the sequence offormula 2: (A1-P2-P3- . . . Pn), with “A” referring to an anionicresidue, “P” referring to a polar residue, and “n” referring to aninteger between about 3 and about 25. Suitable anionic residues includethose known as such in the art, including, without limitation, aspartateand glutamate. Suitable polar residues include those known as such inthe art, including, without limitation, glutamine, glycine, histidine,serine, and threonine. In some aspects, n is an integer such as thatincluding, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25. Preferably, n is an integer such as 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15. In some aspects, the linker sequencehas at least 70% sequence identity to a sequence of formula 2. In someaspects, the amino acid sequence has at least 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence offormula 2. Preferably, the linker sequence has at least 85% sequenceidentity to a sequence of formula 2.

In some aspects, the linker sequence has at least 70% sequence identityto a sequence provided in SEQ ID NO: 34-39. In some aspects, the aminoacid sequence has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to at least one sequence provided inSEQ ID NO: 34-39. Preferably, the linker sequence has at least 85%sequence identity to at least one sequence provided in SEQ ID NO: 34-39.

The antimicrobial agents of the disclosure also include nucleic acidmolecules encoding the amino acid sequences described herein, as well asexpression vectors, encoding an antimicrobial agent, variant, orfragment thereof. Also, the disclosure provides isolated cellscontaining such nucleic acids, expression vectors, or peptides describedherein. The disclosure provides compositions containing nucleic acidmolecules or peptides of the disclosure, as well as variants orfragments thereof. Further, the disclosure provides articles ofmanufacture containing nucleic acid molecules or peptides of thedisclosure, as well as variants or fragments thereof.

The disclosure provides antimicrobial agents having broad antimicrobialactivity against microorganisms including bacteria, yeast, protozoa,fungi, mold, viruses, and combinations thereof. In some aspects, theantimicrobial agents have antimicrobial activity against Gram-positiveand Gram-negative bacteria. The antimicrobial agents of the disclosuremay be used to inhibit or prevent growth of a microorganism. Suchmicroorganisms may include, without limitation, strains of Pseudomonas,Escherichia, Staphylococcus, Streptococcus, Enterococcus, Mycobacteria,Haemophilus, and combinations thereof.

In some aspects, the antimicrobial compositions may include apharmaceutical carrier. In some aspects, the antimicrobial compositionsmay include a food additive.

The disclosure provides methods of reducing or inhibiting growth orsurvival of a microbe. Such methods include contacting the microbe withthe antimicrobial agent.

In some aspects, the disclosure provides a method of reducing orinhibiting growth or survival of a microbe in a subject. The methodincludes contacting, or administering to, the subject a compositioncontaining an antimicrobial agent. A therapeutically effective amount ofthe composition is delivered to the subject.

In some aspects, the disclosure provides a method of treating amicrobial infection in a subject. The method includes contacting, oradministering to, the subject a composition containing an antimicrobialagent disclosed herein. A therapeutically effective amount of thecomposition is delivered to the subject.

In some aspects, the disclosure provides a method of treating mastitisin a subject. The method includes identifying a subject having mastitisand contacting, or administering to, the subject a compositioncontaining an antimicrobial agent. In some aspects, a therapeuticallyeffective amount of the composition is administered directly to themammary tissue of the subject.

The present disclosure further provides an article of manufacture, orkit, that includes a vessel and an antimicrobial agent. The article ofmanufacture may include packaging material that contains theantimicrobial agent and a label that indicates the antimicrobial agentcan be used for treating mastitis. The article of manufacture mayinclude packaging material that contains the antimicrobial agent and alabel that indicates the antimicrobial agent can be used for treating amicrobial infection in a subject. The article of manufacture may includepackaging material that contains the antimicrobial agent and a labelthat indicates the antimicrobial agent can be used for inhibiting thegrowth or survival of microbes. The article of manufacture may includepackaging material that contains the antimicrobial agent and a labelthat indicates the antimicrobial agent can be used for inhibiting thegrowth or survival of microbes in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the disclosure. Thedisclosure may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 graphically illustrates the amino acid composition of someantimicrobial agent aspects. Residue colors reflect a preference forlipophilic (yellow) or basic (blue).

FIG. 2 shows the antimicrobially active dimer structure based onmolecular dynamics simulations. Green and yellow objects are twocompositionally identical peptides oriented in antiparallel alignment.Numbers identify the specific amino acid positions including the aminoterminus (position 0) and the carboxy terminus (position 15). The solidsurface represents the lipid membrane colored according to neutral(blue) or anionic (red) lipid.

FIG. 3 graphically illustrates the roles for specific amino acidpositions within the noncovalent dimer construct for some antimicrobialagent aspects. The illustration shows intradimer stabilizations (blackarrows), dimer-membrane lipid interactions (yellow arrows) anddimer-membrane electrostatic coupling (red-blue arrows).

FIG. 4 graphically illustrates a minimally-spanned covalently-linkeddimer based on the non-covalent formulation shown in FIG. 1. The dimer(silver cartoon and CPK-colored sticks) is shown on a model membranesurface, colored by neutral (blue) vs. anionic (red) lipids.

FIG. 5 graphically illustrates the antibiotic activity of theantimicrobial peptides of the disclosure. A statistically significantdifference (P<0.001) was found between the groups (box-plot below), withthe post-hoc test showing a difference between the WB-14F versus thethree control groups (PBS, ATBX, and Non-Tx) at a 5% level ofsignificance.

FIG. 6 depicts an antimicrobial agent (SEQ ID NO: 17) rendered as sticks(left; as shown above the helical axis) and solvent-accessible surface(center and right, as shown from front and side views, with the helicalaxis pointing up in the plane of the page). Coloration is as follows:hydrophobic residues are yellow; polar residues are colored by element(H=cyan, C=white, N=blue, 0=red).

FIG. 7 shows a membrane disruption model. Toroidal pore formation (FIG.7A) in which the peptide lipophilic surface coordinates the lipid mediumin a systematic way, so as to collaboratively form a transmembrane pore,or in-plane diffusion (FIG. 7B). Individual peptides or dimers locallydisrupt the membrane order via a combination of lipophilicpeptide-membrane interactions and cationic attraction of the negativelycharged phospholipid head groups (red surfaces) by positively chargedpeptide amino acids (blue surfaces), with the local pockets of disorderdiffusively aggregating to form areas of significant membrane weakness.

DETAILED DESCRIPTION

In accordance with the present disclosure, compositions and methods ofusing antimicrobial agents to inhibit the growth or survival ofmicroorganisms have been discovered. In particular, antimicrobial agentshaving peptides with antimicrobial activity are used to inhibit thegrowth or survival of microorganisms. Therefore, the compositions andmethods described herein are useful for antimicrobial treatments andtherapeutics. Furthermore, the compositions and methods described hereinmay be used where antimicrobial resistance has reduced the availabilityof antimicrobial treatments or therapeutics.

I. Compositions

The compositions of the disclosure include antimicrobial agents havingantimicrobial activity against at least one microorganism. In someaspects, the compositions of the disclosure include antimicrobial agentshaving antimicrobial activity against more than one microorganism.

a. Nucleic Acids Encoding Antimicrobial Agents

The disclosure provides antimicrobial agents that include syntheticpeptides having antimicrobial activity. In some aspects theantimicrobial agents include peptides, a fragment thereof, or a variantthereof, as well as nucleic acid molecules that encode the syntheticpeptide of the disclosure. Such nucleic acid molecules may include anexpression vector.

Nucleic acids encoding antimicrobial peptides derived from water buffalohost defense proteins are disclosed. The nucleic acid sequences encodingantimicrobial peptides of the disclosure were derived from SEQ ID NO: 1and the encoded amino acid sequence of SEQ ID NO: 2, having theantimicrobial peptide sequence of SEQ ID NO: 3. Suitable nucleotidesequences of the present disclosure include those that encode a peptidehaving antibacterial activity such as those peptides provided by SEQ IDNOs: 3-33, 40-4101, and those described herein.

Mutant nucleotides of the antimicrobial peptides may be used, so long asmutants include nucleic acid sequences that encode functionalantimicrobial peptides as described herein. The subject nucleic acidsmay be mutated to alter properties of the encoded peptide such asexpression properties, folding properties, solubility properties, andantibacterial activity. A skilled artisan will recognize that proteinsencoded by nucleic acids encoding homologues or mutants may have thesame antibacterial properties as the peptides of SEQ ID NOs: 3-33 and40-4101, or may have altered antibacterial properties. The DNA sequenceor protein product of such a mutation will usually be substantiallysimilar to the sequences provided herein and will differ by one or morenucleotides or amino acids. The sequence changes may be substitutions,insertions, deletions, or a combination thereof. Techniques formutagenesis of cloned genes are known in the art. Methods for sitespecific mutagenesis may be found in Gustin et al., Biotechniques 14:22,1993; Barany, Gene 37:111-23, 1985; Colicelli et al., Mol. Gen. Genet.199:537-9, 1985; and Sambrook et al., Molecular Cloning: A LaboratoryManual, CSH Press 1989, pp. 15.3-15.108 and all incorporated herein byreference. Such mutated nucleic acid derivatives may be used to studystructure-function relationships of a particular antimicrobial peptide,or to alter properties of the peptide that affect its function orregulation. In summary, the disclosure relates to antimicrobial peptidecoding sequences such as those that encode amino acid sequences of SEQID NOs: 3-33, 40-4101, and variants or mutants thereof. Also, thedisclosure encompasses the intermediary RNAs encoded by the describednucleic acid sequences and that translates into an antimicrobial peptideof the disclosure.

b. Antimicrobial Peptide Compositions

A synthetic peptide disclosed herein may be obtained using methods knownin the art, including isolation from a cell expressing the peptide,chemical synthesis, or the peptide may be expressed from a recombinantnucleic acid molecule. Methods of synthesizing a subject peptide aredescribed in Current Protocols in Protein Science, Units 5, pub. JohnWiley & Sons, Inc., 2002 and Current Protocols in Protein Science, Units6, pub. John Wiley & Sons, Inc., 2002 and both are incorporated hereinby reference.

The disclosure contemplates antimicrobial peptides and mutants thereof,which include those peptides encoded by the subject nucleic acids. Thesynthetic antibacterial peptides, or agents, disclosed herein areexemplified by the sequences of SEQ ID NOs: 3-33 and 40-4101. Further,the disclosure includes both homodimers and heterodimers of theantibacterial peptides described herein.

Homologs or peptides that vary in sequence from the amino acid sequencesSEQ ID NOs: 3-33 and 40-4101 are also included in the disclosure. Byhomolog is meant a protein having at least about 10%, usually at leastabout 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or higher amino acid sequence identity to the proteins encodedby SEQ ID NOs: 3-33 or 40-4101, as determined using MegAlign, DNAstar(1998) clustal algorithm as described in Higgins, D. G. and Sharp, P.M., Fast and Sensitive Multiple Sequence Alignments on a Microcomputer,CABIOS, 5: 151-153, 1989, both incorporated herein by reference.

Antimicrobial peptides disclosed herein may be mutated, or altered, toenhance, or change, biological properties of the protein. Suchbiological properties include, but are not limited to, in vivo or invitro stability (e.g., half-life), solubility, amphiphilicity andvarious aspects of antimicrobial activity. Such antimicrobial activityaspects include, without limitation, tissue specificity, microbialtarget selectivity, broad spectrum antimicrobial potency and invarianceas a function of microbial resistance mechanisms. Suitable mutationsinclude single amino acid changes, deletions of one or more amino acids,N-terminal truncations, C-terminal truncations, insertions, etc. Mutantscan be generated using standard techniques of molecular biology,including random mutagenesis and targeted mutagenesis as described inCurrent Protocols in Molecular Biology, Unit 8, pub, John Wiley & Sons,Inc., 2000 and incorporated herein by reference.

The manifold of currently considered peptide formulations is ofsubstantial value because the manifold as a whole has a mechanism ofaction that is not susceptible to a key microbial antioxidativeresistance mechanism conferred by membrane expression ofstaphyloxanthin. Furthermore, strategic sequence variation within themanifold may be applied to counteract microbial defenses effected byadaptive membrane charge distribution corresponding to covalentmodification of anionic lipids, or greater membrane staphyloxanthinexpression.

The antimicrobial peptides disclosed herein are characterized by havingantimicrobial activity. Specifically, the peptides disclosed are capableof disrupting the cell membrane of microorganisms, leading to themicroorganism's destruction.

The antimicrobial peptides generally have a first synthetic amino acidsequence of formula 1 (Gly-X₁-X₂-X₃-X₁-X₁-X₁-Arg-X₄-X₁-X₅-X₆-X₆-Gly)where X₁ is selected from the group of Leu or Ile; X₂ is selected fromthe group of Ala, Val, Leu, Ile, or Pro; X₃ is selected from the groupof Arg or Trp; X₄ is selected from the group of Trp, Ile, or Leu; X₅ isselected from the group of Phe or Trp; and, X₆ is selected from thegroup of Phe, Trp, or Arg.

The antimicrobial peptides also may have a second antimicrobialsynthetic amino acid sequence of formula 1(Gly-X₁-X₂-X₃-X₁-X₁-X₁-Arg-X₄-X₁-X₅-X₆-X₆-Gly) where X₁ is selected fromthe group of Leu or Ile; X₂ is selected from the group of Ala, Val, Leu,Ile, or Pro; X₃ is selected from the group of Arg or Trp; X₄ is selectedfrom the group of Trp, Ile, or Leu; X₅ is selected from the group of Pheor Trp; and, X₆ is selected from the group of Phe, Trp, or Arg. Theantimicrobial agent may include a homodimer of antimicrobial peptides,where a first antimicrobial synthetic sequence has the same sequence asa second antimicrobial synthetic sequence. The antimicrobial agent mayinclude a heterodimer of antimicrobial peptides, where a firstantimicrobial synthetic sequence has a different sequence as a secondantimicrobial synthetic sequence. Such difference may be by one or moreamino acid sequences.

The antimicrobial peptides typically range in length from about 14 aminoacid residues to about 30 amino acid residues. In some aspects, theantimicrobial peptides are about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45 or more amino acid residues in length.Preferably, the antimicrobial peptides are 14 amino acid residues inlength. In some aspects, antimicrobial agents including homodimers ofthe antimicrobial peptides are about 28 amino acid residues in length.In some aspects, antimicrobial agents including homodimers of theantimicrobial peptides are about 35 amino acid residues in length. Insome aspects, antimicrobial agents including heterodimers of theantimicrobial peptides are about 28 amino acid residues in length. Insome aspects, antimicrobial agents including heterodimers of theantimicrobial peptides are about 35 amino acid residues in length. Askilled artisan will recognize that the length of a homodimer orheterodimer of the antimicrobial peptides depends upon the length of theantimicrobial peptide used in the respective homodimer or heterodimer.

The antimicrobial agent may include a linker sequence to connect a firstantimicrobial synthetic sequence to a second antimicrobial syntheticsequence. In some aspects, the linker sequence connected to a first andsecond antimicrobial peptide produces a covalently linked dimerformulation. Suitable linker sequences may be used to improveantimicrobial kinetics. In one aspect, the linker sequence enhanceskinetics by encouraging the dimerized structure to remain in aconformation conducive to antimicrobial activity. In one aspect, thelinker sequence may be used to enhance delivery of the dimer. Suchlinker sequences may include specific amino acid formulations that favorsolubility in polar or lipophilic media. In another aspect, the linkersequence may be used to enhance chemical stability within differentenvironments.

Suitable linker sequences may be tailored to provide specificenhancements. A linker sequence may be a length of any size that doesnot disrupt the antimicrobial activity of the antimicrobial peptide. Insome aspects, the linker sequence may be a length of about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50 or more amino acid residues. Preferably, the linker sequence is alength of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. Insome aspects, the linker sequence is a length of about 7 amino acidresidues. The amino acid residues of the linker sequence may be tailoredto provide specific enhancements. In some aspects, the linker sequenceincludes mostly polar amino acids. In some aspects, the linker sequenceincludes at least one sequence of SEQ ID NO: 34-39. In some aspects, thelinker sequence includes the sequence of SEQ ID NO: 34.

c. Pharmaceutical Compositions

The present disclosure provides a pharmaceutical composition includingat least one antimicrobial agent described herein. To prepare such apharmaceutical composition, an antimicrobial agent, an antimicrobialpeptide, homodimer, heterodimer, or combinations thereof is synthesizedor otherwise obtained, purified as necessary or desired and thenlyophilized and stabilized. The antimicrobial agent may then be adjustedto the appropriate concentration and combined with other agents orpharmaceutically acceptable carriers. Suitable pharmaceuticallyacceptable carriers include, without limitation, a carrier, diluent,excipient, salt, or combinations thereof, that are compatible with theother ingredients of the formulation and not deleterious to therecipient thereof.

Pharmaceutical formulations containing an antimicrobial agent disclosedherein may be prepared by procedures known in the art using well-knownand readily available ingredients. For example, the antimicrobial agentmay be formulated with common excipients, diluents, or carriers, andformed into tablets, capsules, solutions, suspensions, powders, aerosolsand the like. Examples of excipients, diluents, and carriers that aresuitable for such formulations include buffers, as well as fillers andextenders such as starch, cellulose, sugars, mannitol, and silicicderivatives. Binding agents may also be included such as carboxymethylcellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose andother cellulose derivatives, alginates, gelatin, andpolyvinyl-pyrrolidone.

II. Methods

The compositions of the present disclosure including at least oneantimicrobial agent are useful in methods of reducing the growth orsurvival of microorganisms. A skilled artisan will appreciate thenumerous methods of using the antimicrobial agents disclosed herein.Accordingly, the methods included herein are examples and are notlimiting of the scope by which the antimicrobial agents may be used. Themethods of the disclosure include reducing the growth or survival ofmicroorganisms, treating a subject having a microorganism infection, andtreating a subject having mastitis, as well as other methods using theantimicrobial agents disclosed herein.

Microorganisms against which an antimicrobial agent disclosed herein isactive include Gram-negative bacteria, Gram-positive bacteria, yeast,fungi, mold, viruses, unicellular parasites, and other microorganismsknown in the art. Non-limiting examples include Staphylococcus aureus,Listeria monocytogenes, Escherichia coli, Klebsiella pneumoniae,Salmonella typhimurium, Pseudomonas aeruginosa, Proteus mirabilis,Salmonella enteritidis, Neisseria gonorrhoeae, Ureaplasmacanigenitalium, Ureaplasma urealyticum, Candida albicans, Trichomonasvaginalis, Treponema pallidum, Chlamydia trachomatis, Staphylococcushyicus, Coagulase negative staphylococci, Streptococcus agalactiae,Streptococcus hyicus, Streptococcus epidermidis, Streptococcus xylosus,Streptococcus intermedius. Streptococcus dysgalactiae, Streptococcusuberis, Mycoplasma spp. (Mycoplasma bovis, Mycoplasma bovigenitalium,Mycoplasma canadense, Mycoplasma califomicum, Mycoplasma alkalescens),Escherichia coli, Klebsiella spp. (Klebsiella pneumoniae, Klebsiellaoxytoca), Enterobacter spp. (E. aerogenes, E. amnigenus, E. agglomerans,E. arachidis, E. asburiae, E. cancerogenous, E. cloacae, E. cowanii, E.dissolvens, E. gergoviae, E. helveticus, E. hormaechei, E. intermedius,E. kobei, E. ludwigii, E. mori, E. nimipressuralis, E. oryzae, E.pulveris, E. pyrinus, E. radicincitans, E. taylorae, E. turicensis, E.sakazakii, Enterobacter soli), Citrobacter spp (C. amalonaticus, C.braakii, C. diversus, C. farmeri, C. freundii, C. gillenii, C. koseri,C. murliniae, C. rodentium, C. sedlakii, C. werkmanii, C. youngae),Proteus spp. (P. hauseri, P. mirabilis, P. myxofaciens, P. penneri, P.vulgaris), Enterococci, Serratia spp., Pseudomonas spp. (P. aeruginosa,P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P.citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P.oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea), P.chlororaphis group (P. agarici, P. asplenii, P. aurantiaca, P.aureofaciens, P. chlororaphis, P. corrugata, P. fragi, P. lundensis, P.taetrolens), P. fluorescens group (P. antarctica, P. azotoformans, P.brassicacearum, P. brenneri, P. cedrina, P. corrugata, P. fluorescens,P. gessardii, P. libanensis, P. mandelii, P. marginalis, P.mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P.panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P.thivervalensis, P. tolaasii, P. veronii), P. pertucinogena group (P.denitrificans, P. pertucinogena), P. putida group (P. cremoricolorata,P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva, P.plecoglossicida, P. putida), P. stutzeri group (P. balearica P. luteola,P. stutzeri), P. syringae group (P. amygdali, P. avellanae, P.caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P.meliae, P. savastanoi, P. syringae, P. viridiflava), incertae sedis (P.abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P.alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P.cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P.delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P.fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P.jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P.lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana,P. papaveris, P. pelf, P. perolens, P. poae, P. pohangensis, P.protegens, P. psychrophila, P. psychrotolerans, P. rathonis, P.reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P.salomonii, P. segitis, P. septica, P. simiae, P. suis, P.thermotolerans, P. tremae, P. trivialis, P. turbinellae, P.tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P.xanthomarina), Prototheca, Corynebacterium bovis, Arcanobacteriumpyogenes, Bacillus spp. (B. alcalophilus, B. alvei, B. aminovorans, B.amyloliquefaciens, B. aneurinolyticus, B. anthracis, B. aquaemaris, B.atrophaeus, B. azotoformans, B. badius, B. boroniphilus, B. brevis, B.caldolyticus, B. centrosporus, B. cereus, B. circulans, B. coagulans, B.fastidious, B. firmus, B. flavothermus, B. fusiformis, B. galliciensis,B. globigii, B. globisporus, B. infernus, B. insolitus, B. larvae, B.laterosporus, B. lentimorbus, B. lentus, B. licheniformis, B. macerans,B. macquariensis, B. marinus, B. megaterium, B. mesentericus, B.mucilaginosus, B. mycoides, B. natto, B. pantothenticus, B. pasteurii,B. polymyxa, B. popilliae, B. pseudoanthracis, B. pumilus, B.schlegelii, B. sphaericus, B. sporothermodurans, B. stearothermophilus,B. subtilis, B. thuringiensis, B. vulgatis, B. weihenstephanensis),Pasteurella spp. (P. aerogenes, P. anatis, P. avium, P. bettyae, P.caballi, P. canis, P. dagmatis, P. gallicida, P. gallinarum, P.granulomatis, P. langaaensis, P. lymphangitidis, P. mairii, P.multocida, P. oralis, P. pneumotropica, P. skyensis, P. stomatis, P.testudinis, P. trehalosi, P. ureae, P. volantium), Serratia spp. (S.entomophila, S. ficaria, S. fonticola, S. grimesii, S. liquefaciens, S.marcescens, S. odorifera, S. plymuthica, S. proteamaculans, S.quinivorans, S. rubidaea, S. symbiotica), and other microorganisms knownin the art. Such unicellular parasites include, without limitation,leishmania, treponema, giardia, and others known in the art.

In some aspects, microorganisms against which an antimicrobial agentdisclosed herein is active may also include medically relevant bacteria.Such medically relevant bacteria may include, without limitation,Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. intermedius,S. lugdunensis, S. saprophiticus, S. schleiferi, S. warneri;Streptococcus agalactiae, S. anginosus, S. bovis, S. equisimilis, S.mitis, S. mutans, S. pneumonia, S. pyogenes, S. sanguis, S. salivarius,S. suis; Enterococcus faecalis, E. faecium; Neisseria gonorrhoeae, N.meningitides; Moraxella catarrhalis, M. lacunata, M. nonliquefaciens, M.urethralis; Kingella kingae, K. denitrificans; Eikenella corrodens;Bacillus alvei, B. anthracis, B. brevis, B. cereus, B. circulans, B.coagulans, B. licheniformis, B. macerans, B. pumilus, B. sphaericus, B.subtilis, B. thuringiensis; Clostridium botulinum, C. bifermentans, C.butyricum, C. camis, C. clostridioforme, C. difficile, C. fallax, C.histolyticum, C. innocuum, C. perfringens, C. novyi, C. ramosum, C.septicum, C. sordellii, C. tertium, C. tetani; Listeria monocytogenes;Erysipelothrix rhusiopathiae; Propionibacterium acnes, Streptobacillusmoniliformis; Calymmatobacterium granulomatis; Bacteroides distasonis,B. fragilis, B. thetaiotaomicron, B. ovatus, B. vulgatus; Fusobacteriummortiferum, F. nucleatum, F. necrophorum; Gemella morbillorum;Peptostreptococcus (P. anaerobius, P. asaccharolyticus, P. hydrogenalis,P. magnus, P. melaninogenica, P. micros, P. tetradius, P. prevotii);Porphyromonas (P. asaccharolytica, P. gingivalis); Prevotella (P.melaninogenica, P. oris, P. buccae); Veillonella parvula; Salmonellacholeraesuis, S. enteritidis, S. gastroenteritis, S. paratyphi-A, S.schottmuelleri, S. typhi, S. typhimurium; Shigella boydii, S.dysenteriae, S. flexneri, S. sonnei; Arcobacter cryaerophilia, A.butzleri, A. skirrowii; Campylobacter coli, C. fetus, C.hyointestinalis, C. jejuni, C. lari, C. sputorum, C. upsaliensis;Helicobacter pylori, H. cinaedi, H. fennelliae; Vibrio cholera, V.damsela, V. fetus, V. fluvialis, V. furnissia, V. hollisae, V. mimicus,V. parahaemolyticus, V. vulnificus; Citrobacter freundii, C. diversus(C. koseri); Escherichia coli; Enterobacter aerogenes, E. cloacae;Klebsiella pneumoniae, K. rhinoscleromatis, K. ozaenae; Proteusmirabilis; Serratia marcescens; Pseudomonas aeruginosa, P. cepacia, P.fluorescens, P. maltophilia, P, mallei, P. pseudomallei, P. putida, P.putrefaciens, P. stutzeri; Brucella abortus, B. canis, B. melitensis, B.suis; Pasteurella haemolytica, P. multicida; Yersinia enterocolitica, Y.pestis, Y. pseudotuberculosis; Francisella tularensis; Haemophilusinfluenza, H. parainfluenzae, H. ducreyi, H. aegyptius; Bordetellaavium, B. bronchiseptica, B. hinzii, B. parapertussis, B. pertussis;Corynebacterium diphtheria, C. kutscheri, C. pseudotuberculosis, C.pseudodiphtheriticum, C. renale, C. xerosis, C. ulcerans; Mycobacteriumafricanum, M. avium-intracellulare, M. bovis, M. fortuitum, M. kansasii,M. leprae, M. marinum, M. microtii, M. tuberculosis, M. scrofulaceum, M.ulcerans; Nocardia asteroides, N. brasiliensis, N. caviae; Actinomycesbernardiae, A. bovis, A. denticolens, A. gerencseriae, A. georgiae, A.hordeovulneris, A. howellii, A. hyovaginalis, A. israelii, A. meyeri, A.naeslundii, A. neuii, A. odontolyticus, A. pyogenes, A. radingae, A.slackii, A. suis, A. turicensis, A. viscosus; Propionibacteriumpropionicus; Streptomyces somaliensis; Leptospira interrogans, L.biflexa, Borrelia burgdorferi; B. hermsii, B. recurrentis, B. turicatae;Spirillum minum; Treponema pallidum, T. carateum; Mycoplasma fermentans,M. genitalium, M. hominis, M. penetrans, M. pirum, M. pneumonia;Ureaplasma urealyticum; Rickettsiae akari, R. australis, R. conorii, R.japonica, R. prowazekii, R. rickettsii, R. sibirica, R. typhi; Ehrlichiacanis, E. chaffeensis, E. equi, E. ewingii, E. phagocytophila, E.platys, E. risticii, E. sennetsu; Orientia tsutsugamushi; Coxiellaburnetii; Bartonella bacilliformis; B. henselae, B. Quintana; Chlamydiapneumonia, C. psittaci, C. trachomatis; Legionella ansia, L. dumollii,L. feelei, L. micdadei, L. pneumophila, and other medically relevantbacteria known in the art or yet to be discovered.

In some aspects, microorganisms against which an antimicrobial agentdisclosed herein is active may also include veterinary relevantbacteria. Such relevant bacteria may include, without limitation,Acholeplasma laidlawii; Acinetobacter Iwoffii; Actinobacillus equuli, A.lignieresii, A. pleuropneumoniae, A. rossi, A. suis; Actinobaculum suis;Actinomyces bovis, A. viscosus; Aeromonas hydrophila, A. salmonicida;Aliivibrio salmonicida; Anaplasma phagocytophilum; Arcobacter butzleri;Avibacterium paragallinarum; Bacillus anthracis, B. cereus, B.licheniformis, B. subtilis, B. thuringiensis; Bacteroides fragilis;Bartonella clarridgeiae, B. elizabethae, B. henselae, B. vinsonii;Bibersteinia trehalosi; B. avium, B. bronchiseptica, B. parapertussis,B. pertussis; Borrelia afzelii, B. anserine, B. burgdorferi, B. garinii;Brachyspira aalborgi, B. alvinipulli, B. hyodysenteriae, B. intermedia,B. pilosicoli, B. suanatina; Brochothrix thermosphacta; Brucellaabortus, B. canis, B. ceti, B. melitensis, B. ovis, B. pinnipedialis, B.suis; Burkholderia mallei, B. pseudomallei; Campylobacter coli, C.fetus, C. jejuni, C. lad, C. upsaliensis; Chlamydia trachomatis;Chlamydophila abortus, C. felis, C. psittaci; Citrobacter freundii;Clostridium botulinum, group I, II, III, IV; Clostridium chauvoei, C.difficile, C. haemolyticum, C. novyi, C. perfringens, C. piliforme, C.septicum, C. sordellii, C. tetani; Corynebacterium bovis, C. diphtheria,C. kutscheri, C. pseudotuberculosis, C. renale, C. ulcerans; Coxiellaburnetii; Dermatophilus congolensis; Dichelobacter nodosus; Edwardsiellaictaluri, E. tarda; Ehrlichia canis, E. ruminantium; Enterococcusfaecalis, E. faecium; Erysipelothrix rhusiopathiae; Escherichia coli;Flavobacterium columnare, F. psychrophilum; Francisella noatunensis, F.tularensis; Fusobacterium canifelinum, F. equinum, F. necrophorum;Gallibacterium anatis; Haemophilus parasuis; Helicobacter hepaticus, H.pylori; Histophilus somni; Klebsiella oxytoca, K. pneumoniae;Lactobacillus plantarum; Lawsonia intracellularis; Legionellapneumophila; Leptospira borgpetersenii, L. interrogans, L. kirschneri;Listeria innocu, L. ivanovii, L. monocytogenes; Listonella anguillarum;Mannheimia granulomatis, M. haemolytica, M. varigen; Melissococcusplutonius; Microcystis aeruginosa; Moraxella bovis, M. osloensis;Morganella morganii; Mycobacterium avium, M. bovis, M. leprae, M.marinum, M. tuberculosis; Mycoplasma agalactiae, M. bovis, M.capricolum, M. felis, M. gallisepticu, M. haemofelis, M. hyopneumoniae,M. hyorhinis, M. hyosynoviae, M. meleagridi, M. mycoides, M. pneumonia,M. pulmonis, M. suis, M. synoviae; Neisseria gonorrhoeae; Neorickettsiaristicii; Nicoletella semolina; Nocardia asteroids; Paenibacilluslarvae; Pasteurella caballi, P. dagmatis, P. multocida, P.pneumotropica; Peptoniphilus indolicus; Plesiomonas shigelloides;Prevotella melaninogenica; Proteus mirabilis, P. vulgaris; Pseudomonasaeruginosa, P. anguilliseptica; Renibacterium salmoninarum; Rhodococcusequi; Rickettsia prowazekii, R. rickettsia; Riemerella anatipestifer;Salmonella spp., S. enterica; Serratia marcescens; Shigella dysenteriae,S. flexneri; Staphylococcus aureus, S. epidermidis, S. felis, S. hyicus,S. intermedius, S. pseudintermedius, S. schleiferi; Stenotrophomonasmaltophilia; Streptobacillus moniliformis; Streptococcus agalactiae; S.canis, S. devriesei, S. dysgalactiae, S. equi, S. pneumonia, S.porcinus, S. pyogenes, S. suis, S. uberis; Taylorella asinigenitalis, T.equigenitalis; Treponema pallidum, T. paraluiscuniculi, T. pedis, T.phagedenis; Trueperella pyogenes; Ureaplasma diversum; Vibrioalginolyticus, V. cholera, V. parahaemolyticus, V. vulnificus; Yersiniaenterocolitica, Y. pestis, Y. pseudotuberculosis, Y. ruckeri and otherveterinary relevant bacteria known in the art or yet to be discovered.

The method of reducing the growth or survival of microorganisms includescontacting a microorganism population with an antimicrobial agent of thepresent disclosure. The method may include first identifying amicroorganism population.

The methods disclosed herein include methods of inhibiting the survivalor growth of a microorganism. The term “inhibit” includes a decrease inany detectable amount, for example, 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or more, and can be determined by methods known in theart. Exemplary methods of detecting inhibition includes a determinationby count, such as a bacterial culture or viral titer, or by evaluationof one or more symptoms associated with infection by a microorganism.Symptoms associated with infection by a microorganism are known in theart. Such symptoms are characteristic to a particular infectiousmicroorganism and the resulting condition.

A composition that can support growth or survival of a microorganism maybe contacted with an effective amount of the antimicrobial agent in avariety of ways. For example, if the composition is a food item, theantimicrobial agent may be added directly to the food, it may beincorporated into the matrix of the packaging material or it may becoated onto the packaging material, in which case the antimicrobialagent may be released during storage, upon dissolution of theencapsulation material, contact with moisture or at a predeterminedtemperature. If the composition is a body fluid, the antimicrobial agentmay be added directly to the body fluid.

A subject may be contacted with the antimicrobial agent in a variety ofways including, without limitation, administration using routes commonlyknown in the art. Such routes include oral, parenteral (includingsubcutaneous, intravenous, intramuscular and intraperitoneal), rectal,vaginal, dermal, transdermal (topical), transmucosal, intrathoracic,intrapulmonary and intranasal (respiratory) routes. The means ofadministration may be by injection, using a pump or any otherappropriate mechanism.

An antimicrobial agent disclosed herein may be administered to a subjectin a single does, in multiple doses, in a continuous or intermittentmanner, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled artisans. Theadministration of the antimicrobial agents may be essentially continuousover a pre-selected period of time or may be in a series of spaceddoses. Both local and systemic administration is contemplated.

The dosage to be administered to a subject may be any amount appropriateto reduce or prevent infection or to treat at least one symptomassociated with the infection. Some factors that determine appropriatedosages are well known to those skilled in the art and may be addressedwith routine experimentation. For example, determination of thephysicochemical, toxicological and pharmacokinetic properties may bemade using standard chemical and biological assays and through the useof mathematical modeling techniques known in the chemical,pharmacological and toxicological arts. The therapeutic utility anddosing regimen may be extrapolated from the results of such techniquesand through the use of appropriate pharmacokinetic and pharmacodynamicmodels. Other factors will depend on individual patient parametersincluding age, physical condition, size, weight, the condition beingtreated, the severity of the condition, and any concurrent treatment.The dosage will also depend on the agent chosen and whether preventionor treatment is to be achieved, and if the agent is chemically modified.

The precise amount to be administered to a subject will be theresponsibility of the attending physician. However, to achieve thedesired effects, an antimicrobial agent disclosed herein may beadministered as single or divided dosages. For example, of at leastabout 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kgto about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight,although other dosages may provide beneficial results.

The absolute weight of a given antimicrobial agent disclosed hereinincluded in a unit dose may vary widely. For example, about 0.01 toabout 2 g, or about 0.1 to about 500 mg, of at least one antimicrobialagent may be administered. Alternatively, the unit dosage may vary fromabout 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g toabout 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2g.

Daily doses of the antimicrobial agents may vary as well. Such dailydoses may range, for example, from about 0.1 g/day to about 50 g/day,from about 0.01 g/day to about 25 g/day, from about 0.1 g/day to about12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day toabout 4 g/day, and from about 0.5 g/day to about 2 g/day.

An antimicrobial agent may be used alone or in combination with a secondmedicament. The second medicament may be a known antimicrobial agentsuch as, but not limited to, a β-lactam, macrolide or other antibiotics,e.g. Azithromycin, Doxycycline, Tetracycline, and Erythromycin; anantifungal agent such as clotrimazole, nystatin, fluconazole,ketoconazole, amphotericin B, caspofungin, or voriconazole; an agenteffective against a protozoan such as, for example, Metronidazole ortimidazole. The second medicament may also be an antiviral agent such asAbacavir, Acyclovir, Amantadine, Didanosine, Emtricitabine, Enfuvirtide,Entecavir, Ganciclovir, Gardasil, Lamivudine, Nevirapine, Nelfinavir,Oseltamivir, Ribavirin, Rimantadine, Ritonavir, Stavudine, Valaciclovir,Vidarabine, Zalcitabine, and Zidovudine. The effective amount of thesecond medicament will follow the recommendations of the secondmedicament manufacturer, the judgment of the attending physician andwill be guided by protocols and administrative factors for amounts anddosing as indicated in the PHYSICIAN'S DESK REFERENCE (as commonly knownin the art).

The effectiveness of the method of treatment may be assessed bymonitoring the subject for signs or symptoms of the microbial infectionas discussed above, as well as determining the presence or amount ofmicroorganism present in the subject by methods known in the art.

III. Kits

The present disclosure provides articles of manufacture and kitscontaining materials useful for treating the conditions describedherein. The article of manufacture may include a container of acomposition as described herein with a label. Suitable containersinclude, for example, bottles, vials, and test tubes. The containers maybe formed from a variety of materials such as glass or plastic. Thecontainer holds a composition having an antimicrobial agent disclosedherein, which is effective for inhibiting the growth of a microorganismor treating a condition caused by a microorganism. The label on thecontainer may indicate that the composition is useful for treatingspecific conditions and may also indicate directions for administration.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications are incorporated by reference in their entirety. In theevent that there is a plurality of definitions for a term herein, thosein this section prevail unless stated otherwise.

As used herein, “administering” is used in its broadest sense to meancontacting a subject with a composition disclosed herein.

As used herein, the term “antimicrobial activity” means microbicidal ormicrobiostatic activity or a combination thereof, against one or moremicroorganisms. Microbicidal activity refers to the ability to kill orcause irreversible damage to a target microorganism. Microbiostaticactivity refers to the ability to inhibit the growth or proliferativeability of a target microorganism without necessarily killing orirreversibly damaging it.

As used herein, the term “identity” or “sequence identity” refers to arelationship between two or more polypeptide sequences, as well as twoor more polynucleotide sequences, namely a reference sequence and agiven sequence to be compared with the reference sequence. Sequenceidentity is determined by comparing the given sequence to the referencesequence after the sequences have been optimally aligned to produce thehighest degree of sequence similarity, as determined by the matchbetween strings of such sequences. Upon such alignment, sequenceidentity is ascertained on a position-by-position basis. For example,the sequences are “identical” at a particular position if at thatposition, the nucleotides or amino acid residues are identical. Thetotal number of such position identities is then divided by the totalnumber of nucleotides or residues in the reference sequence to givepercent (%) sequence identity. Sequence identity can be readilycalculated by known methods, including but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. N., et., OxfordUniversity Press, New York (1988), Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York (1993); ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinge, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York(1991); and Carillo et al., Applied Math., 48:1073 (1988), the teachingsof which are incorporated herein by reference.

As used herein, the term “inhibit” means a decrease in any amountincluding, without limitation, a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%.

The term “nucleic acid” is used herein to refer to a polymer ofdeoxynucleic ribose nucleic acids, as well as ribose nucleic acids. Theterm includes linear molecules, as well as covalently closed circularmolecules. It includes single stranded molecules, as well as doublestranded molecules.

As used herein, “subject” refers to a living organism having a centralnervous system. In particular, subjects include, but are not limited to,human subjects or patients and companion animals. Exemplary companionanimals may include domesticated mammals (e.g., dogs, cats, horses),mammals with significant commercial value (e.g., dairy cows, beefcattle, sporting animals), mammals with significant scientific values(e.g., captive or free specimens of endangered species), or mammals thatotherwise have value. Suitable subjects also include: mice, rats, dogs,cats, ungulates such as cattle, swine, sheep, horses, and goats,lagomorphs such as rabbits and hares, other rodents, and primates suchas monkeys and apes. In some aspects, subjects may be diagnosed with amicrobial infection, may be at risk for a microbial infection, or may beexperiencing a microbial infection. Subjects may be of any age includingnew born, adolescence, adult, middle age, or elderly.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to increase to some beneficial degree, preferably toincrease by at least about 1 to 100 percent, more preferably by at leastabout 5 to 95 percent, and more preferably by at least 8 percent orhigher, healing or infection improvement as compared to untreatedcontrols. An “effective amount” is a pharmaceutically-effective amountthat is intended to qualify the amount of an agent or compound, thatwhen administered to a subject, will achieve the goal of healing aninfection site, inhibiting the growth of a microorganism, or otherwisebenefiting the recipient environment.

The term “variant” relates to nucleotide or amino acid sequences whichhave similar sequences and that function in the same way.

Amino acid designations may include full name, three-letter, orsingle-letter designations as commonly understood by one of ordinaryskill in the art to which this disclosure belongs.

As various changes could be made in the above compositions and methodswithout departing from the scope of the disclosure, it is intended thatall matter contained in the above description and in the Examples givenbelow, shall be interpreted as illustrative and not in a limiting sense.

EXAMPLES Example 1 Antimicrobial Activity of Antimicrobial Agents

The antimicrobial agents disclosed are derivatives of SEQ. ID. NO.: 3,having 14 amino acids and a broad spectrum of antimicrobial activity.The antimicrobial agents of the disclosure were synthesized and analyzedto evaluate antimicrobial activity. Initially, the antimicrobial agentof SEQ ID NO.: 3 was evaluated for inhibition of clinical isolates ofcommon mastitis pathogens (Table 1).

TABLE 1 Antimicrobial activity of the antimicrobial agent of SEQ ID NO.:3 expressed as minimum inhibitory concentration (MIC). Clinical pathogenMIC isolate (μM) Bacteria strain 335 B-S 12.5 Streptococcus dysgalactiae335 B-15 25.0 Klebsiella pneumoniae 404 C-18 12.5 Corynebacteriumspecies 416 B-10 25.0 Staphylococcus aureus 331 C-10 25.0 Staphylococcusaureus 422 C-10 25.0 Staphylococcus aureus

Additional antimicrobial agents were evaluated for antimicrobialactivity, including 23 computationally-derived analogues of SEQ ID NO:3. These analogues were grouped into two functional series, includingthose synthesized for improved helicity (Table 2; A-P) and thosesynthesized for enhanced activity (Table 2; IND, Q-T, and W-X). Theantimicrobial activity of the synthetic analogues provided in Table 2were evaluated using standard techniques such as those described inSang, et al. 2007 Dev. Comp. Immunol., and herein incorporated byreference. Based on the data collected, three synthetic antimicrobialagents were identified (Table 2, shaded rows) as having superiorantimicrobial activity compared to the agent of SEQ ID NO: 3 or SEQ IDNO: 19 (Table 2).

TABLE 2

Example 2 Structural/Function Analysis of Antimicrobial Agents

The structure/function mechanisms for the antimicrobial agents describedherein were evaluated. In order to develop a better understanding of theeffects of different amino acids at different positions as well asdeveloping the most biologically active and stable synthetic peptide(s), Quantitative Structure-Activity Relationship (QSAR) modeling wasused to guide the optimization of multi-drug resistant Staphyloccocusaureus antimicrobial formulations.

Among the four main mechanisms for cellular peptidic antimicrobialaction, two (the barrel-stave and toroidal pore) models require stableamphiphilic helical peptides, and a third (in-plane diffusion) requiressignificant intramolecular stabilization as may be achieved by secondary(helices or sheets) or tertiary (disulphide bonds or salt bridges)structural elements that can guarantee preservation of peptidicamphiphilicity. For peptide families that are known to possessantimicrobial activity and have structural aspects indicative of helixformation (i.e., few or no prolines; sequence with polar-nonpolaralternating in period of about 4 monomers), it is reasonable to assumethat the bioactive conformation of the peptide is helical, and that themechanism of action is one of the three listed above. From thisassumption, the antimicrobial agents provided herein have been tailoredto have a high helical propensity (as predicted via the Agadir software)and to display substantial amphiphilicity in helical conformation. Suchattributes are depicted in FIG. 5 for one of the most potentantimicrobial agents described herein, SEQ ID NO: 17.

The classic signature of the barrel-stave model is a periodicalternation of hydrophobic, anionic, hydrophobic and cationic patches,enabling the peptides to electrostatically self-assemble in cellularmembranes into stable pores that exclude the surrounding lipids andcompromise membrane integrity. The antimicrobial agents described hereinare strictly cationic, thus obviating the barrel-stave model since agiven peptide generally associates more preferentially with the lipidmedium than with other peptides. However, they may still significantlyinterfere with the membrane via either of the remaining two possiblemodels depicted in FIG. 7.

The high degrees of helical stability and amphiphilicity evident in thepeptide formulations strongly suggests either the formation of toroidalpores which span microbe cell membranes inducing cell ion leakage (FIG.7A), or else the formation of dimers whose selective lipid interactionsdisrupt the geometric regularity and stability of the lipid membrane,where these interaction include peptide cation interactions with lipidanions, and peptide aryl disruption of lipid aliphatic/aliphaticinteractions.

Based on the examples described herein, the antimicrobially-potentpeptide formulations based primarily on improving helical propensity andamphiphilicity profiles suggest that the observed activity arises fromone of the above two mechanisms. The highly systematic nature of thepeptides (rigorously hydrophobic, except for arginines in every fourthor fifth position) argues for the more structurally regular model of atoroidal pore (which could functionally assume the role of avoltage-gated channel to compromise cellular pH as per the KvAPpeptide). However, the short length of antimicrobial agent peptides (˜21Å from end-to-end in helical conformation) implies that they may not belong enough to span the membrane, which would argue for the in-planediffusion model.

Some very basic qualitative rules to explain observed antimicrobialactivity trends have been resolved.

-   -   1) Given Trp in sequence position 4, peptide activity is        proportional to the steric compactness of the lipophilic residue        at position 3 (i.e., Pro>Ala>Val>Leu), which suggests that        spatial compactness is optimal toward the N-terminus of the        peptide,    -   2) If position 4 has anything other than a large lipophile, the        backbone kink induced by proline is intolerable at position 3,        and proline is not tolerated at all at any other position,    -   3) Given Arg in position 4, peptide activity varies according to        Ala-3>Val-3>Leu-3>Pro-3, which is to say that spatial        compactness is desirable, but the backbone kink induced by        proline is unacceptable in this case, and    -   4) Interchanging residues at positions 12 and 13 does not have a        major effect on activity, but significant changes to only one of        these residues kills activity, indicating that the        lipophilic/electropositive balance in this region is critical to        activity.

To differentiate between the two mechanisms of action paradigmsdescribed in the previous section, one strategy would be to selectivelysubstitute aromatic residues (i.e., Phe, Trp) for aliphatic (Ile, Leu)and vice versa. These substitutions should generally preserve theamphiphilicity that is required in both models, and some substitutionsmay enhance helical stability (which also favors both models), howevertheir effect may vary as follows:

-   -   a) Aryl groups, being planar, are more compact than the branched        alkyl groups in Ile and Leu side chains and stack better with        the planar guanine groups in the requisite arginines, which may        favor the formation of orderly pores that interface well with        the membrane, while    -   b) Alkyl groups are inherently more attractive to the        predominantly alkyl lipid medium, but their branched nature        tends to produce more structural disorder as per the in-plane        diffusion scenario.

Having elucidated some important structure/function trends, Agadircalculations on conservative substitutions (i.e., ones that change thesize and electrostatic character of a residue a little but not much)were performed to further optimize the antimicrobial activity of the WBsynthetic peptides. Therefore, the peptides listed below (SEQ ID NO:26-33) were synthesized and evaluated to capitalize on their helicalstability (Table 3).

TABLE 3 Antimicrobial agents optimized for helical stability. PeptideSequence WB14-F1 (SEQ ID NO: 26) GLARILLRLLFFRG WB14-N1 (SEQ ID NO: 27)GLARILLRLLFRFG WB14-O1 (SEQ ID NO: 28) GLVRILLRLLFRFGWB14-O2 (SEQ ID NO: 29) GLVRILLRLLLRFG WB14-O3 (SEQ ID NO: 30)SLVRILLRLLLRFG WB14-O4 (SEQ ID NO: 31) GLTRILLRLLLRFGWB14-O5 (SEQ ID NO: 32) GLVRILLRLLLRYG WB14-O6 (SEQ ID NO: 33)GLVRILLRLLLRFS

Example 3 Antimicrobial Activity of Antimicrobial Agent Analogs

The additional antimicrobial agent analogs optimized for helicalstability in Example 2 (Table 3) were evaluated for antimicrobialactivity. The antimicrobial activity of the synthetic analogues providedin Table 3 were evaluated using standard techniques such as thosedescribed in Sang, et al. 2007 Dev. Comp. Immunol., and hereinincorporated by reference. The antimicrobial activity of the agents isprovided in Table 4.

TABLE 4

The results indicated that the antimicrobial agent having SEQ ID NO: 8was the most efficacious antimicrobial synthetic peptide. Theantimicrobial activity of SEQ ID NO. 8 was further evaluated againstclinical isolates of mastitis-causing bacteria (Table 5).

TABLE 5 In vitro antimicrobial activity of the WB-14F peptide (SEQ IDNO.: 8) against clinical isolates of mastitis-causing bacteria. ClinicalMultidrug MIC Isolate Bacterial strain resistance (μg/ml) 335-B05Streptococcus dysgalactiae ++ 8.0 335-B15 Klebsiella pneumoniae ++++16.0 404-C18 Corynebacterium species ++ 16.0 416-B10 Staphylococcusaureus +++ 2.0 212-B05 Escherichia coli ++ 4.0 BAA 44* (ATCC)Staphylococcus aureus +++++ 4.0 The WB-14F was synthesized under thefollowing conditions: >95% purity; TFA salt; All were L-amino acids; Noterminal modification; *ATCC cat. No. BAA 44.

Example 4 In Vivo Analysis of Antimicrobial Activity

The antimicrobial agent of SEQ ID NO: 8 was evaluated using an in vivomodel of bacterial infection to assess the antimicrobial activity of theagent. The following methods and materials were used.

Bacterial Strain.

Staphylococcus aureus was isolated from milk from dairy cows withclinical mastitis (Livestock Disease Diagnostic Center, University ofKentucky. Lexington, Ky.). Upon arrival, bacteria were striped on bloodagar plates (TSA+5% sheep blood from Remel, Kansas City. Mo.) andcultured at 37° C. for 24 hrs. A fresh single bacterial colony pickedfrom the agar plate was inoculated into 10 ml of Muller Hinton II broth(MHB) and cultured at 37° C. for 4-6 hrs. until reaching an absorbanceof 0.8 with a 625 nm spectrophotometer filter. At this absorbance, theMHB has been documented to contain 1×10⁵ colony forming units (CFU) in a50 volume.

Synthesis of the Water Buffalo Host Defense Peptide.

The WB-14F peptide (SEQ ID NO: 8; GLARILLRWLFFRG) was chemicallysynthesized (Peptide2.0; Chantilly, Va.). After synthesis, the WB-14Fmolecule eluted in a single peak on RP-HPLC, and confirmed by massspectroscopy. Final purity of the peptide was >97% with a molecularweight of 1718.4. The peptide was lyophilized and stored at −80° C.until use.

Mastitis Mouse Model.

For the experiment, 56 female Balb/c mice (BW˜50 g) at 7-10 days oflactation were used. Four groups of n=14 mice were used for eachexperiment as described below. For intramammary inoculation the protocoldescribed by Chandler was followed (J. Med. Microbiol. V3; 273-282:1970) with minor modifications. Briefly, lactating mothers wereseparated from the pups 4 hours before inoculation. Then, pups wereallowed to lactate for 1 hour before inoculation to empty the mammaryglands. Pups were separated again from the mother and never reunitedwith her afterwards. After that, mice were anesthetized with acombination of Tiletamine/Zolazepam (Zoletil® 100, Virbac) at a dose of100 mg/kg IM, and the mammary glands were disinfected with 70% alcohol.Using a stereoscopic microscope a 50 μL of the previously prepared S.aureus suspension (containing 1×10⁵ CFU) was inoculated intra-mammarily.Injections were made slowly, using a 33 G needle (Hamilton, Kent, UK),into the left and right abdominal teats (L4 and R4). After inoculationmice were allowed to recover from the anesthesia.

Therapeutic Effect of the WB-14F Compared to Controls and AntibioticTreated Groups.

Mice (n=14/group) were treated intra-mammarily using the stereoscopictechnique as follows: Group 1: WB-14F (32 μg diluted in 50 μl sterilePBS per gland); Group 2: Control, Sterile Phosphate Buffered Saline (50μl per gland); Group 3: Ceptiofur (400 μg diluted in 50 μl mineral oilper gland); Group 4: infected but untreated. All groups (except Group 4)received the treatments 24 hours post-infection every 24 hrs. for 3consecutive days (total of 3 treatments).

Determination of Colony Forming Units (CFU) in Mammary Glands.

All mice were euthanized (using CO₂) following the AVMA euthanasiaguidelines 24 hours after the last treatment. The infected mammaryglands (L4 and R4) were removed individually using soft tissuedissection and homogenized in 1 ml of sterile PBS in Falcon tubes usinga Polytron tissue homogenizer (Kinematica, Switzerland). Dilutions(10⁻¹, 10⁻², and 10⁻³) of the homogenates were plated quantitatively todetermine the number of CFU per gland. Results were reported as theaverage of the results from the two glands (L4 and R4) from each animal.

Statistical Analysis.

Kruskal-Wallis with Dunn's post-test was performed using GraphPad PrismVer. 5.0a for Mac, GraphPad Software, San Diego Calif. USA. Astatistically significant difference (P<0.001) was found between thegroups (FIG. 5), with the post-hoc test showing a difference between theWB-14F versus the three control groups (PBS, ATBX, and N1901.27on-Tx) ata 5% level of significance.

Example 5 Structural/Functional Analysis of Linked Dimer PeptideAntimicrobial Agents

A key component of the preliminary design and screening ofcovalently-linker peptide dimer formulations entailed the use ofmolecular dynamics simulations.

The sequence-specific activity dependence observed from in vivo studiesis closely corroborated by intradimer interactions observed in moleculardynamics simulations of unlinked (non-covalent) peptide dimers.Subsequent molecular dynamics simulations have produced strong evidencethat the interactions evident in the non-covalent dimers (showngraphically in FIG. 3) are preserved or amplified inspecially-formulated covalently-linked dimers (as shown in FIG. 4).

Key structural features of the non-covalent and covalently-linked dimersystems that best corroborate experimental in vivo activities include 1)preservation of monomer helical structure, 2) retention of intradimerlipophilic and electrostatic coupling, plus strong evidence of both ofthe following intermolecular interactions: 3) peptide/membrane nonpolarcoupling whereby peptide aryl amino acids interact with membrane lipidsso as to insert aryl groups between individual lipid molecules, and 4)peptide/membrane electrostatic coupling between peptide cationicresidues and anionic lipid head groups. For each of these fourinteraction classes, specific quantitative metrics were defined based onthe relative percentage of simulation time in which specific atoms ofthe peptide reside within set distances of specific atoms of theintramolecular or intermolecular interaction partners. Specifically,consistent preservation of non-polar interatomic distances of no morethan 4.0 Angstroms were used to identify preservation of key nonpolarfeatures, and consistent preservation of polar interatomic distances ofno more than 2.5 Angstroms were used to identify preservation of keypolar features.

The four structural features described above were found via moleculardynamics simulations to be particularly well preserved or amplified bythe following specific covalent linker formulations: DNNNNNN (SEQ ID NO:34); DGGGGGG (SEQ ID NO: 38); DNNNNN (SEQ ID NO: 35); and, DGGGGG (SEQID NO: 37), where in each of the above cases the N-terminus of linkersequence covalently couples via a normal peptide bond to the C-terminusof the first peptide monomer, and the C-terminus of the linker sequencecovalently couples via a normal peptide bond to the N-terminus of thesecond peptide monomer.

The invention illustratively disclosed herein suitably may be practicedin the absence of any element, which is not specifically disclosedherein. It is apparent to those skilled in the art, however, that manychanges, variations, modifications, other uses, and applications to themethod are possible, and also changes, variations, modifications, otheruses, and applications which do not depart from the spirit and scope ofthe disclosure are deemed to be covered by the disclosure, which islimited only by the claims which follow.

1. An antibiotic peptide comprising a synthetic amino acid sequencehaving the sequence of formula 1Gly-X₁-X₂-X₃-X₁-X₁-X₁-Arg-X₄-X₁-X₅-X₆-X₆-Gly

wherein X₁ is selected from the group of Leu or Ile, X₂ is selected fromthe group of Ala, Val, Leu, or Ile, X₃ is selected from the group of Argor Trp, X₄ is selected from the group of Trp, Ile, or Leu X₅ is selectedfrom the group of Phe or Trp, and X₆ is selected from the group of Phe,Trp, or Arg.
 2. The antimicrobial composition of claim 1, wherein thecomposition inhibits growth of bacteria.
 3. (canceled)
 4. Theantimicrobial composition of claim 2, wherein the bacteria are selectedfrom the group consisting of Pseudomonas, Escherichia, Staphylococcus,Streptococcus, Enterococcus, Mycobacteria, Haemophilus, and combinationsthereof.
 5. The antimicrobial composition of claim 1 further comprisinga pharmaceutical carrier.
 6. The antimicrobial composition of claim 1,wherein the synthetic amino acid sequence has at least 85% sequenceidentity to SEQ ID NO:
 8. 7. A method for inhibiting growth of bacteriacomprising contacting the bacteria, for a time and under conditionseffective to inhibit bacterial growth, with a composition comprising asynthetic amino acid sequence having the sequence of formula 1Gly-X₁-X₂-X₃-X₁-X₁-X₁-Arg-X₄-X₁-X₅-X₆-X₆-Gly

wherein X₁ is selected from the group of Leu or Ile, X₂ is selected fromthe group of Ala, Val, Leu, or Ile, X₃ is selected from the group of Argor Trp, X₄ is selected from the group of Trp, Ile, or Leu X₅ is selectedfrom the group of Phe or Trp, and X₆ is selected from the group of Phe,Trp, or Arg.
 8. (canceled)
 9. The method of claim 7, wherein thebacteria are selected from the group consisting of Pseudomonas,Escherichia, Staphylococcus, Streptococcus, Enterococcus, Mycobacteria,Haemophilus, and combinations thereof.
 10. The method of claim 89,wherein the bacteria are selected from the group consisting ofStreptococcus agalactiae, Staphylococcus aureus, Enterobacter aerogenes,Klebsiella pneumoniae, Actinomyces pyogenes, Streptococcus uberis,Streptococcus dysgalactiae, and combinations thereof.
 11. The method ofclaim 7, wherein the growth of bacteria is inhibited at least 10%.
 12. Amethod of treating microbial infection, the method comprising: a.identifying a subject having a microbial infection; and, b.administering a therapeutically effective amount of an antibioticcomposition to the subject, wherein the antibiotic composition comprisesa synthetic peptide having the sequence of formula 1:Gly-X₁-X₂-X₃-X₁-X₁-X₁-Arg-X₄-X₁-X₅-X₆-X₆-Gly

wherein X₁ is selected from the group of Leu or Ile, X₂ is selected fromthe group of Ala, Val, Leu, or Ile, X₃ is selected from the group of Argor Trp, X₄ is selected from the group of Trp, Ile, or Leu X₅ is selectedfrom the group of Phe or Trp, and X₆ is selected from the group of Phe,Trp, or Arg.
 13. The method of claim 12, wherein the antibioticcomposition further comprises a pharmaceutical carrier.
 14. The methodof claim 12 further comprising inhibiting the growth of a mastitisinfection causing microorganism by at least 10%.
 15. A method oftreating mastitis, the method comprising: a. identifying a subjecthaving mastitis; and, b. administering a therapeutically effectiveamount of an antibiotic composition to the subject, wherein theantibiotic composition comprises a synthetic peptide having the sequenceof formula 1: Gly-X₁-X₂-X₃-X₁-X₁-X₁-Arg-X₄-X₁-X₅-X₆-X₆-Gly

wherein X₁ is selected from the group of Leu or Ile, X₂ is selected fromthe group of Ala, Val, Leu, or Ile, X₃ is selected from the group of Argor Trp, X₄ is selected from the group of Trp, Ile, or Leu X₅ is selectedfrom the group of Phe or Trp, and X₆ is selected from the group of Phe,Trp, or Arg.
 16. The method of claim 15, wherein the antibioticcomposition is administered to the mammary tissue of the subject. 17.The method of claim 15 further comprising inhibiting the growth of amastitis infection causing microorganism by at least 10%.